1. Field of the Invention
The invention provides prophylactic, therapeutic and industrial antioxidant compositions that are enhanced with stabilized atomic hydrogen/free electrons. The invention also provides methods to prepare and use such antioxidant compositions.
2. Description of the Related Art
Molecular oxygen is an essential substance for all aerobic organisms, including humans. Oxygen is involved in many metabolic reactions ranging from energy production to the synthesis of vitamin A and prostaglandins and the deoxification and metabolism of drugs, chemicals and foods. Some forms of oxygen and oxygen-containing species are very reactive and can cause significant damage to the organism. Such moieties are termed reactive oxygen species (xe2x80x9cROSxe2x80x9d).
ROS include hydrogen peroxide, hydroxyl radical, superoxide radical, singlet oxygen, etc. Hydrogen peroxide is relatively stable and remains until it is destroyed or reacts with molecules sensitive to oxidative damage. Other ROS, such as hydroxyl radicals, are very unstable and last no longer than a few picoseconds to seconds, depending on the environment. The hydroxyl radical is one example of another reactive group, referred to as free radical species. Free radicals are atoms, ions or molecules that contain an unpaired free electron. The presence of an unpaired free electron is one of the reasons for the high reactivity and short lifetime of most such species. Free radicals and ROS are normal products of metabolism and are actually involved in the regulation of cellular processes [C. K. Sen and L. Packer, FASEB J., Vol. 10, 227 (1996)]. However, the overproduction of ROS and free radicals is involved in the pathogenesis of a wide variety of human diseases [C. K. Sen and L. Packer, FASEB J., Vol. 10, 227 (1996)]. Such diseases include cancer, diabetes, AIDS, cardiovascular diseases, neurodegenerative diseases, skin diseases, autoimmune diseases and others.
ROS can damage biological macromolecules, cells, tissues and organs in many ways. Oxidation of sulfhydryl groups and other sensitive components of proteins can either increase or decrease the activity of enzymes. It was also recently discovered that oxidative modification of proteins is involved in the control and regulation of many cellular processes [C. K. Sen and L. Packer, FASEB J., Vol. 10, 227 (1996)]. Peroxidation of membrane lipids can result in crosslinking of unsaturated lipids and modification of the cellular permeability to different ions and molecules. Some ions such as calcium are also involved in the control and regulation of cellular processes. Hydroxylation of nucleic acid bases and the breakup of nucleic acids are also deleterious processes that result from the presence of excessive concentrations of ROS. Once formed, many free radicals are involved in xe2x80x9cchain reactionsxe2x80x9d producing other free radicals and ROS. Even the scavenging and degradation of free radicals can produce highly reactive, damaging species. [E. R. Stadtman, Science, Vol. 257, 1220 (1992)].
Organisms, including humans, have evolved ways of handling dangerous ROS. Organisms posses a large number of defenses against the deleterious effects of ROS. [See, for instance, Oxidative Stress, Oxidants and Antioxidants, Academic Press, London, 1991]. Many enzymes and small molecules are used by aerobic cells to protect against the damage caused by ROS. Enzymes which are used to catalyze the removal or transformation of ROS include superoxide dismutase, catalase, glutathione peroxidase, glutathione transferase etc. Small molecules used by aerobic cells to scavenge ROS include vitamins C, E and A, glutathione, ubiquinone, uric acid, carotenoids, etc. Superoxide dismutase is a very efficient catalyst for the removal of superoxide free radicals. Such removal results in the production of hydrogen peroxide. Catalase, in turn, is a very efficient catalyst for the removal of the hydrogen peroxide that is produced. Glutathione peroxidase and transferase enzymes are efficient in the removal of many ROS. Among small molecules, the most important molecule involved in the prevention of damage caused by ROS is a thiol-containing tripeptide, glutathione [see, for instance, Oxidative Stress, Oxidants and Antioxidants, Academic Press, London, 1991]. Glutathione (GSH) is present in all animal cells in millimolar concentrations and is directly involved in the reduction (and, thereby, detoxification) of ROS. Reduction of ROS by glutathione results in oxidation and dimerization of glutathione to the disulfide-linked dimer (GSSG). This oxidized form of glutathione is toxic and oxidizing in itself. Other small molecules such as ascorbate (vitamin C) or tocopherol (vitamin E) also can directly reduce ROS. The difference between enzymes such as superoxide dismutase and small molecules such as vitamin C is that the former can catalytically remove many molecules of ROS, while the latter reacts with oxidants stoichiometrically, usually in a 1:1 or 2:1 ratio.
However, ROS and free radicals are not always toxic. Recent evidence suggests that at moderately high concentrations, certain forms of ROS such as hydrogen peroxide, may act as signal transduction messengers involved in the control of cell proliferation, differentiation and death [C. K. Sen and L. Packer, FASEB J., Vol. 10, 227 (1996)]. Regulation of gene expression by different concentrations of many oxidants and antioxidants has recently been shown. It was shown that the activity of many proteins involved in signal transduction and gene transcription is modified by intracellular redox state [C. K. Sen and L. Packer, FASEB J., Vol. 10, 227 (1996)]. In fact, the regulation of gene expression by oxidants, antioxidants, and the cellular redox state has emerged as a novel subdiscipline in molecular biology that has promising therapeutic implications [C. K. Sen and L. Packer, FASEB J., Vol. 10, 227 (1996)]. Redox-regulated transcription factors such as AP-1 and NFxcexaB have been shown to be implicated in the pathogenesis of many inflammatory diseases, cancer, AIDS, diabetes complications, atherosclerosis and neurodegenerative diseases. It was observed that critical steps in the signal transduction cascades are sensitive to oxidants and antioxidants. It was also shown that the interaction of some membrane proteins, protein phosphorylation and the binding and activation of transcription factors are sensitive to physiological oxidant-antioxidant homeostasis [C. K. Sen and L. Packer, FASEB J., Vol. 10, 227 (1996)]. Sen and Packer suggested those oxidants, antioxidants and other factors that influence intracellular redox status can be developed into novel potential prophylactic and therapeutic agents.
Scientists and other individuals involved in the development of pharmaceutical and dietetic products based on antioxidant action realized the potential usefulness of antioxidants long ago. Many products with potential therapeutic use are described in peer reviewed journals and patent literature. However, those products are currently not completely satisfactory. We will discuss such prior art literature below.
Small molecules are currently used as dietetic supplement antioxidants. Vitamins C and E are probably the most commonly used antioxidant supplements. However, both molecules can easily be oxidized and, when oxidized, become toxic themselves [M. Gabbay et al., Neuropharmacology, Vol. 35, 571 (1996)]. Novel synthetic molecules such as 21-aminosteroids, also termed lazaroids, showed some effect in the prevention of free radical damage to tissue after brain damage but did not show any beneficial effects in clinical trials with stroke patients [RANTTAS Investigators, Stroke, Vol. 27, 195 (1996)]. Lipoic acid and its derivatives have been proposed as therapeutic antioxidants, but those molecules rapidly leave cells and do not sufficiently protect affected tissues from oxidative damage [U.S. Pat. No. 5,728,735]. N-acetyl cysteine (NAC) showed similar positive effects to lipoic acid but it also leaves cells very rapidly [U.S. Pat. No. 5,762,922]. Dithiocarboxylates and ditiocarbamates also showed some promise as therapeutic antioxidants [U.S. Pat. No. 5,821,260]. But at higher concentrations these molecules are toxic. Carotenoids have also showed some limited therapeutic effects in various chronic diseases, including coronary heart diseases, cataract and cancer.
Catalytic antioxidant enzymes have also been tested for their potential therapeutic effects in diseases that are related to the overproduction of free radicals and reactive oxygen species [Uyama et al., Free Radic. Biol. Med., Vol. 8, 265 (1990)]. Superoxide dismutase and catalase showed some therapeutic effects in ROS overproduction related diseases such as stroke, heart attack, and autoimmune diseases such as Crohn""s disease and lupus [U.S. Pat. No. 5,834,509, page 6]. The disadvantage of using this approach is that such molecules are large, unstable macromolecules and cannot penetrate in high concentrations to the affected cells and tissues. Moreover, enzymes are proteins and cannot be effectively administered orally. Proteins are digested in the stomach and small intestines and, therefore, most of the catalytic activity is lost. Finally, recombinant proteins are also very expensive.
Small molecules with the catalytic ability to remove superoxide and hydrogen peroxide have recently been synthesized and tested. [U.S. Pat. No. 5,223,538] Such molecules are usually complexes of transition metals such as manganese, copper, zinc, iron or cobalt. Some of the recently tested molecules such as manganese salen complexes [U.S. Pat. No. 5,834,509] even showed catalytic ability to reduce concentrations of both superoxide and hydrogen peroxide. Most of such molecules are either toxic or lack the catalytic ability in-vivo. However, some of the recently tested manganese salen complexes showed low toxicity and reasonable protection against stroke or other tissue degenerative diseases. On the other hand, such molecules cannot scavenge other reactive oxygen species and free radicals such as singlet oxygen, hydroxyl radicals, nitric oxide, peroxynitrite, hypochlorous acid and organic peroxides. [U.S. Pat. No. 5,403,834].
Several other approaches to reduce the toxic effects of ROS and free radicals were tested. Chelating agents were used to prevent the production of very reactive hydroxyl radicals. Such agents bind metals, such as iron or copper, and subsequently prevent the reactions of metal cations with hydrogen peroxide, which yield hydroxyl radicals. Iron chelators and desferroxiamine showed some limited efficiency in the treatment of neurodegenerative diseases. [B. Halliwell, Free Radic. Biol. Med., Vol. 7, 645 (1989)] Unfortunately, at the higher concentrations needed for enhanced activity, such agents are toxic. Spin trapsxe2x80x94a class of molecules which are used to trap free radicals to measure free radical concentration, were also used to prevent the tissue damage caused by ROS. [X. Cao and J. W. Phillis, Brain Res., Vol. 644, 267 (1994)] Unfortunately, at the higher concentrations needed for the greater efficiency of such reagents, unacceptable toxicity appears.
As described in the preceding paragraph, it is clear that more efficient antioxidant agents for prophylactic, therapeutic and industrial uses are needed. Accordingly, an object of the present invention is to provide a novel composition of antioxidants enhanced with stabilized atomic hydrogen having low toxicity. Another object is to provide a method of producing stabilized atomic hydrogen and preparing pharmaceutical and industrial compositions using the stabilized atomic hydrogen. Methods of using such pharmaceutical and industrial compositions and methods of using stabilized atomic hydrogen with other therapeutic agents are also described.
Atomic hydrogen is a reducing free radical consisting of a proton and an electron. It is the second most powerful reducing agent known and it also can release the strongest reducing agent, that is, the free electron [M. Pach and R. Stosser, J. Phys. Chem. A, Vol. 101, 8360 (1997)]. It was recently shown that this very reactive and unstable species can be encaged inside cage-like compounds and become stable for months or even years [R. Sasamori et al., Science, Vol. 265, 1691 (1994)]. In one embodiment of this invention, we describe methods to prepare and stabilize atomic hydrogen photoelectrochemically, electrochemically and plasma-chemically. We also describe the use of the most succesful, biomedically acceptable, cage-like compounds, including cobalamin (vitamin B12), potassium silicate and colloidal silicates and aluminosilicates such as zeolites.
In another embodiment of this invention, we describe the preparation of mixtures of stabilized atomic hydrogen with thiol antioxidants, and other highly efficient antioxidants such as polyphenols. Such mixtures are efficient in keeping the concentration of reduced glutathione in cells constant and, subsequently, in preventing oxidative damage to cells.
In yet another embodiment of the invention, it is described how pharmaceutical compositions enhanced with stabilized atomic hydrogen are used to modify gene expression regulation, cell death, proliferation and differentiation for therapeutic purposes.
Furthermore, it is described how pharmaceutical compositions enhanced with the stabilized atomic hydrogen are used in the treatment of cancer, autoimmune diseases such as, diabetes or arthritis, neurological disorders, neurodegenrative diseases, cardiovascular diseases, skin diseases and other disorders which are related to the overproduction of reactive oxygen species and free radicals.
In another embodiment of this invention, we describe how stabilized encaged atomic hydrogen enhanced antioxidants are used to prevent the oxidation of food, industrial oils, plastics and other oxidation-prone materials.
Based on the foregoing, it is clear that more efficient prophylactic and therapeutic antioxidant agents are needed. Such reagents should be of low toxicity, inexpensive to manufacture and store, stable in-vitro and in-vivo, and reactive towards all or most free radicals and reactive oxygen species. Moreover, such agents should be able to penetrate affected cells and tissues and remain there for a prolonged period of time in active form. The ability to penetrate the blood brain barrier would also be a desirable property. The ability to interact with other therapeutic agents without adverse effects would be an important advantage. Finally, the antioxidant agents which can be used to control gene expression regulation and cell proliferation, death or differentiation would be extremely useful therapeutic agents. It is one object of the invention to provide methods to produce and use such a novel class of antioxidants. Such antioxidants could also be used as dietetic supplements. Moreover, such antioxidants could be used to prevent oxidation and free-radical damage in food and in industrial products such as oils and plastics.
As previously mentioned, aerobic organisms on Earth evolved by acquiring the ability to utilize oxygen. However, products of oxygen metabolism, reactive oxygen species, are toxic to most organisms. As also described earlier, evolving organisms developed numerous enzymes such as superoxide dismutase or use small molecules such as vitamin E to scavenge such oxidizing species. It was recently shown that one of the oldest enzymes that can scavenge reactive oxygen species was actually a hydrogenase: an enzyme that splits molecular hydrogen into atomic hydrogen [R. P. Happe et al., Nature, Vol. 385, 126 (1997)]. Atomic hydrogen is a proton plus an electron and is the second most efficient antioxidant after the free electron itself. However, atomic hydrogen can also release free electrons when it encounters powerful oxidants. Unfortunately, atomic hydrogen is also a very reactive species and it disappears within milliseconds of its production.
While some techniques for stabilizing atomic hydrogen have been developed, the atomic hydrogen produced by these techniques can be stabilized only at very low temperatures in solidified noble gas matrices [S. N. Foner et al., J. Chemical Physics, Vol. 32, 963 (1960)]. Recently, Japanese scientists showed that atomic hydrogen can be encaged into cage-like silicates and stabilized at room temperature in solid state and organic solvents for longer than one year [R. Sasamori et al., Science, Vol. 265, 1691 (1994)]. Moreover, mild oxidants such as oxygen, could not scavenge encaged atomic hydrogen, but stronger oxidants caused a release of free electrons. German scientists recently independently confirmed this unusual and important discovery [M. Pach and R. Stosser, J. Phys. Chem. A, Vol. 101, 8360 (1997)]. Shirahata and coworkers showed that the active component of electrochemically-reduced water is also atomic hydrogen, probably encaged into molecular hydrogen bubbles [S. Shirahata et al., Biochem. Biophys. Res. Comm., Vol. 234, 269 (1997)]. However, organic silicates used by German and Japanese scientists to encage atomic hydrogen are toxic, and large amounts of molecular hydrogen used in Shirahata""s work are toxic such that they are not suitable for pharmaceutical use.
The various features of novelty which characterize the invention are pointed out with particularity in the claims annexed to and forming a part of the disclosure. For a better understanding of the invention, its operating advantages, and specific objects attained by its use, reference should be had to the descriptive matter in which there are illustrated and described preferred embodiments of the invention.